The invention relates to methods for identifying compounds that kill bacteria or inhibit bacterial growth. The invention also relates to methods for identifying compounds that can be used to treat infections (e.g., bacterial infections in organisms such as mammals).
Bacterial cell wall peptidoglycan biosynthesis is a multistep process (see FIG. 1). Although there is some variation between bacterial species, each step in the respective synthetic pathways is essential for the growth of the bacteria. Inhibition of any step can be lethal, and each step is therefore a potential target against which new antibacterial drugs are sought. Inhibitors are already known for some steps in the biosynthetic pathway; however, bacteria have developed resistance to many of these inhibitors, thus necessitating continued searching for new antibacterial agents.
One mode of defense that gram positive bacteria use to resist a certain class of antibacterial agents (i.e., the xcex2-lactams, which inhibit peptidoglycan formation) is to produce an enzyme called xcex2-lactamase. Production of xcex2-lactamase is induced in some bacterial strains by the presence of xcex2-lactams in the cell. xcex2-Lactamase reacts with xcex2-lactam drugs (e.g., penicillin or cephalosporin), rendering the drugs inactive. Certain species of gram negative bacteria such as Enterobacter (e.g., E. cloacae, E. kobei, E. agglomerans, or E. flavus) and Citrobacter freundii also produce xcex2-lactamase, in response to the build-up of cell wall degradation products, not just in the presence of xcex2-lactams per se. Because bacterial cell walls are continuously degraded and reassembled throughout the life cycle of a bacterium, the build-up of degradation products can be due to inhibition of at least one step in the peptidoglycan biosynthetic pathway.
The invention features new assays based on the discovery that induction of the xcex2-lactamase gene can be used to identify compounds that kill bacteria (i.e., bacteriocidal activity) or inhibit bacterial growth (i.e., bacteriostatic activity), and thus to treat bacterial infections (i.e., to reduce symptoms of existing infections and to prevent infections) in organisms such as mammals. The xcex2-lactamase can be encoded, for example, by a xcex2-lactamase gene normally carried by a bacterial host, or inserted into a host, e.g., a heterologous host. The new methods are highly efficient and sensitive, and can be used, for example, for high throughput screening of libraries of potential inhibitors.
In one embodiment, the invention features a method for identifying a candidate compound (e.g., a single compound or a member of a library of potential inhibitors) that inhibits bacterial growth. The method includes the steps of contacting bacteria with the candidate compound to form a reaction mixture, and then assaying the reaction mixture for induction of xcex2-lactamase, which indicates inhibition of bacterial growth.
The assaying step can, for example, include measuring the optical absorbance (e.g., optical density (OD)) of the reaction mixture (e.g., to detect the absorbance of xcex2-lactamase at 490 nm); detecting the binding of antibodies to xcex2-lactamase; or probing for xcex2-lactamase mRNA.
In this context, a xe2x80x9ccandidate compoundxe2x80x9d is any compound not previously known to inhibit xe2x80x9cbacterial growth,xe2x80x9d which includes proliferation of bacteria, budding, cell division, endospore formation, and other forms of reproduction. xe2x80x9cInhibitors of bacterial growthxe2x80x9d include both compounds that prevent growth of bacteria (i.e., bacteriostatic compounds) and compounds that kill bacteria (i.e., bacteriocidal compounds).
The invention also features a method for identifying an inhibitor of cell wall biosynthesis. The method includes the steps of contacting bacteria with a candidate compound to form a reaction mixture; and assaying the reaction mixture for induction of xcex2-lactamase, wherein induction of xcex2-lactamase indicates that the candidate compound is an inhibitor of cell wall biosynthesis.
In another embodiment, the invention features a method for identifying a candidate compound that can be used to treat infection in an organism by a bacteria. The method includes the steps of contacting the bacteria with the candidate compound to form a reaction mixture, and then assaying the reaction mixture for induction of xcex2-lactamase, which indicates that the candidate compound can be used to treat bacterial infection.
Organisms that can be treated include mammals (e.g., humans, non-human primates, horses, cows, pigs, sheep, goats, dogs, and cats); non-mammalian animals (e.g., chickens or frogs); other eukaryotes (e.g., plants); and prokaryotes.
The invention also features a method for identifying a candidate compound that inhibits bacterial growth. The method includes the steps of providing bacteria carrying a gene that encodes xcex2-lactamase; incubating the bacteria with the candidate compound under conditions that enable cell wall biosynthesis to form a reaction mixture; and assaying for induction of xcex2-lactamase, which indicates that the candidate compound is an inhibitor of bacterial growth.
A bacteria xe2x80x9ccarrying a genexe2x80x9d is a bacteria that contains a plasmid, cosmid, vector, or other nucleic acid molecule that includes the gene. The gene can be incorporated into a chromosome (e.g., integrated into a bacterial chromosome) or can be extrachromosomal, but still within the bacterial cell. The gene can be from the same bacterial species as the host, e.g., preexisting in the host, or from a different species (i.e., heterologous). The gene can be a xcex2-lactamase gene from a bacterial species selected from the group of genera consisting of Citrobacter, Enterobacter, Serratia, Pseudomonas, and Proteus. For example, the gene can be, ampC from Citrobacter freundii. The gene can also include a reporter gene such as lacZ or luc. The reporter gene can be fused to the xcex2-lactamase gene or otherwise under the control of the same regulators as xcex2-lactamase.
The method can also include the steps of obtaining a cell extract containing enzymes, cofactors, and carrier molecules necessary for a particular step or steps of cell wall biosynthesis; supplying a substrate for the step or steps; incubating the candidate compound with the cell extract and the substrate under conditions that enable the step or steps to proceed to form an incubation mixture; and assaying the incubation mixture for the substrate and the product produced in the step or steps. The production of an amount of product less than that normally produced in the step or steps relative to the amount of substrate indicates the presence of an inhibitor of the step or steps.
In addition, the invention features a method for identifying an inhibitor of a particular step or steps of cell wall biosynthesis. The method includes the steps of providing bacteria carrying a gene that encodes xcex2-lactamase; incubating the bacteria with a candidate compound under conditions that enable cell wall biosynthesis to form a reaction mixture; assaying the reaction mixture for induction of xcex2-lactamase to identify an inhibitor of cell wall biosynthesis; obtaining a cell extract containing. enzymes, cofactors, and carrier molecules necessary for the particular step or steps; supplying a substrate for the step or steps; incubating the inhibitors with the cell extract and the substrate under conditions that enable the step or steps to proceed; and assaying the incubation mixture (e.g., by chromatography) for the substrate and the product normally produced in the step or steps. The production of an amount of product less than that normally produced in the step or steps relative to the amount of substrate indicates the presence of an inhibitor of the step or steps.
The cell extract can be a whole cell, a cell membrane preparation, or a cytoplasmic extract, for example. The substrate can be detectably labeled (e.g., with a fluorescent tag, an isotopic label, or biotin).
The particular step of cell wall biosynthesis referred to above can be the enolpyruvyl transfer step. catalyzed by MurA or MurZ; the reduction of uridine diphosphate N-acetylenolpyruvylglucosamine catalyzed by MurB; the addition of L-alanine to uridine diphosphate-N-acetylmuramic acid catalyzed by MurC; the addition of D-glutamic acid to uridine diphosphate-N-acetylmuramic acid-L-alanine catalyzed by MurD; the addition of meso-diaminopimelate to uridine diphosphate-N-acetylmuramic acid-dipeptide catalyzed by MurE; the addition of D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid-tripeptide catalyzed by MurF; the racemization of L-alanine to D-alanine catalyzed by Ala racemase; the ligation of two molecules of D-alanine catalyzed by D-Ala:D-Ala ligase; the synthesis of lipid-linked N-acetylmuramic acid-pentapeptide catalyzed by MraY; the N-acetylglucosamine transfer step catalyzed by MurG; septum peptidoglycan synthesis catalyzed by the peptidoglycan transglycosylase-transpeptidase Ftsl; or septum peptidoglycan synthesis catalyzed by FtsW.
An effective amount of a compound identified by these methods as an inhibitor of bacterial growth or as an inhibitor of particular steps in cell wall biosynthesis can be administered to an organism as a method of treating bacterial infection. An xe2x80x9ceffective amountxe2x80x9d of a compound is an amount of the compound that, upon administration to an existing organism, reduces the spread of or completely eradicates a bacterial infection, or that prevents infection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, technical manuals, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The new methods represent a surprising discovery in that compounds that kill or inhibit the growth of bacteria can be identified at levels below their respective minimum inhibitory concentrations (MIC) by assaying for the induction of xcex2-lactamase. Previous screens for bacteriocidal and bacteriostatic compounds looked for inhibition; the present methods are based on induction.
The new methods have numerous advantages. For example, the methods are used to detect induction of xcex2-lactamase, rather than indirectly observing inhibition of xcex2-lactamase by assaying for intact xcex2-lactams or identifying an MIC by looking for absence of growth. As a result, the new methods allow detection of interruptions in steps of the cell wall biosynthesis process other than just those provoked by xcex2-lactams.
Other advantages include efficiency, ease of use, good quantitation, sensitivity (e.g., effective drugs can be detected at concentrations below the MIC), reliability, reproducibility, selectivity, facility, versatility (e.g., the methods are adaptable from benchtop to high throughput screening methodology), and robustness (e.g., the screening methods can use natural product extracts which are very dirty).
The high sensitivity of the new methods can allow detection of certain compounds that are neither bacteriostatic nor bacteriocidal but nonetheless affect cell wall biosynthesis. Although such compounds might not themselves be effective drugs, they can be used to lead to novel drugs. For example, the compounds discovered by any of the new methods can serve as a basis for the design of structural analogs, some of which are likely to be more effective than the initially discovered compounds. The structural analogs can also be screened by the new methods.
Furthermore, the new methods allow screening for inhibitors of reactions, rather than inhibitors of enzymes. This is important for at least two reasons. First, some known inhibitors of cell wall biosynthesis (e.g., vancomycin) bind to the substrate of a reaction thereby rendering that substrate unavailable for reaction with an enzyme. The enzyme itself is not affected by the inhibitor; nonetheless the observed result is the same (i.e., the enzymatic reaction is ceased). Second, multiple steps in cell wall biosynthesis can be carried out by a single, multiple domain enzyme, while certain inhibitors can block the activity of just one of the domains. For example, two of the final transformations in cell wall biosynthesis are mediated by a single enzyme having two domains, only one of which is inactivated by xcex2-lactam antibiotics.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.